Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of AIA sensory motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from OIA ganglia, as well as the methods for establishing and maintaining sensory motor neurons in culture. Hi, I am Aliza from Laboratory of Kelsey Martin at the Department of Psychiatry and the Department of Biological Chemistry at the University California Los Angeles.

Hi, I'm Wan also from the marketing lab. Today I'm going to show you the procedure of carting and sensory motor neurons from Clac Apia Konica. In Our laboratory, we use this procedure to study the cell and the molecular biology of synaps formation and synaptic plasticity.

So let's get started. To begin culturing a pleio neurons first prepare culture dishes by coating the glass well of a matte tech glass bottom culture dish with poly L lysine made in sodium bore. Add enough to completely cover the glass and incubate at room temperature overnight.

After incubation, thoroughly remove the poly L lysine by rinsing in artificial sea water four to five times. After removing the last rinse, add two milliliters of a solution containing half L 15 solution with salts and L-glutamine and half hemolymph. This culture medium must be added to the dish at least one hour prior to plating cells so that the hemolymph sticks to the dish.

Next, clean the tools and cigar dishes with ethanol. Rinse them thoroughly with double distilled water and then place them for at least one hour under a UV light in the culture hood. To prepare sharp electrodes for microdissection of neurons, use a micro electrode polar and 100 millimeter long glass capillaries with a 1.5 millimeter outer diameter and a 0.86 to 1.12 millimeter inner diameter.

Use pulling parameters that produce an electrode with a long and wispy fine tip of such high resistance that there is no capillary intake of fluid. When placed in solution, a fluid meniscus in the electrode will damage the neuron during isolation. Next, prepare the anesthetic solution culture medium and protease solution.

Once ready, use adult apia of about 80 to 100 grams for isolation of sensory neurons and of LFS motor neurons. Use juvenile one to four gram apia for isolation of L seven motor neurons to make culture media a fresh or thawed aliquot of OIA hemolymph is required. Hemolymph can be collected in mass in the spring of each year to supply the lab over the course of the year, select large animals that weigh between 500 grams and one kilogram to extract hemolymph.

To begin swaddle the animal in a disposable underpad so that only a small portion of the animal is exposed. Clean that portion of the skin with ethanol and then hold it while another person uses a sterile razor blade to make an incision in the exposed area. Squeeze the animal so that the hemolymph squirts into a clean beaker.

Be sure that it does not contact the dirty skin of the animal. Repeat this process making an incision at a different location on the same animal. Pool together collections from each individual animal, but do not mix hemo limps collected from different animals.

As this can introduce contaminants. Spin the hemolymph at 2000 times G for 10 minutes to remove blood cells. Aliquot the supernatant in 10 milliliter aliquots and label samples by animal.

These aliquots can be stored at negative 80 degrees Celsius, but not at negative 20 degrees Celsius or a precipitate will form begin dissection of the ganglia by anesthetizing the animals. To do this inject 0.35 molar magnesium chloride into adult apsia for 80 to 100 gram adults use a 60 milliliter syringe with an 18 gauge 1.5 inch needle for one to four gram juveniles. Use a 10 milliliter syringe with a 21 gauge needle.

Enter the animal at an angle of about 35 degrees. Be sure not to enter too deeply to avoid penetrating the internal organs. The animal should become very distended and relaxed.

Next, pin the anesthetized animal to a dissecting dish with a pin at the head and the tail and the foot facing up. 18 gauge needles work well for pinning 80 to 100 gram animals and 21 gauge needles. Work well on one to four gram animals.

Hold the skin of the neck with tooth forceps and cut through the skin in underlying connective tissue along the full length of the foot from head to tail with the surgical scissors. Pin the two sides down, then using forceps and scissors. Cut the esophagus and pull it to the side to expose the ganglia using a pair of fine forceps and find scissors, cut out the ganglia.

Leave a fair amount of nerve since this makes it easier to pin down the ganglia. After protease treatment to digest the ganglia, place them in protease solution. Then incubate them at 34 to 35 degrees Celsius.

80 to 100 gram animals should be digested for 135 minutes and one to four gram animals for 105 minutes. Increase the incubation time to allow easier de sheathing. Decrease the incubation time to prevent proteolytic damage of the neurons after incubation.

Use the fine tip forceps to transfer and wash the ganglia in artificial seawater three times. Keep the ganglia in L 15 medium to de sheath the ganglia. Transfer the protease digested ganglia to a cigar coated 60 millimeter or 35 millimeter dish containing L 15.

The ganglia is now ready to be dissected under a stereo microscope with external halogen. Remove the petal ganglia from the pleural ganglia. Now pin down the pleural ganglia in the proper orientation using insect pins and a fine forceps with the fine forceps and Vana surgical scissors.

Carefully lift the connective tissue and cut it to expose the sensory cluster. Be careful not to damage the neuronal cell bodies. Next, remove any excess connective tissue from the dish and add more medium.

Making sure to never expose the des sheath ganglion to air. Now proceed with removing the neurons of interest for culture. The sensory cluster consists of approximately 200 clustered neurons of 40 to 50 micron diameter.

Set a pin to make a post at a short distance from the ganglion. Next, isolate the neurons using a long, sharp electrode. Pull out the identified neurons one by one by touching the cell body just off center.

Make sure that you do not impale the neuron. Then slowly and steadily, pull the cell soma together with the axon away from the ganglion. Gently tap the electrode against the pin to dislodge neurons from the electrode.

Next, use a disposable pipette tip to transfer individual neurons from the sigar dish to a culture dish to prevent the neurons from sticking to the plastic pipette tip. Aspirate the culture medium through the tip before it is used to transfer neurons. Take great care to avoid exposing the neurons to any air bubbles or to any extreme forces.

Distribute the neurons evenly throughout the dish. Then use a sharp electrode to gently straighten the neurons processes and tap them down to the bottom of the dish. Next, leave the dish on the microscope stage to incubate at room temperature for no less than three hours.Overnight.

Incubation is routine. Cover the stage with aluminum foil to protect it from light. Make sure that the microscope stage is unperturbed during this period.

After the room temperature incubation, gently transfer the dish to an 18 degree Celsius incubator for continued growth. After three hours at 18 degrees Celsius, culture should adhere to the dish and after six to 12 hours, new neurite growth should be visible. Begin by isolating the LFS motor neurons.

Pin the ganglion so that the ventral surface is facing up. Using forceps, pull the connective tissue sheath back to expose the entire half of the ganglion containing the LFS motor neurons and de sheath the remaining parts of the ganglion. Remove any connective tissue from the dish and add more L 15.

Then remove the LFS motor neurons. It helps to notice that the LFS neurons are differentiated from neighboring L EENs neurons on the basis of a small dark pigment spot in their sam. To isolate the L seven or L 11 motor neuron, pin the ganglion so that the dorsal surface is facing up.

Using forceps, pull the connective tissue sheath back to expose the half of the ganglion containing both L seven and L 11. Be careful not to ever touch or damage the neuronal. So place a pin to make a post at a short distance from the ganglion.

The L seven neuron can be differentiated from the L 11 neuron by three criteria. First, the L 11 is cod to L seven. Second, the L 11 cell body is more oblong in shape.

And third, the L 11 axon tends to branch close to the cell body isolate neurons by removing neurons with a sharp electrode as described for the sensory neurons. Now isolate, transfer and spread out neurons on the culture dish as previously described. To prepare the sensory motor co-culture, use the glass electrode to coax the sensory axon to come into contact with the motor neuron.

Do this very gently using the side of the tapered electrode to finally have the sensory axon physically contact the axon of the motor neuron. Do not lift the culture dish, but rather gently glide the dish along the microscope stage. Follow the incubation procedure previously described with the same anticipated growth rate.Eight.

We'll just show you how to make a application sunscreen motor neuron. When Doing this procedure, it's important to remember to leave the cultures on the microscope stage undisturbed overnight before moving them into the incubator. That's it.

Thanks for your watching and Good luck for your experiments.